New methods for the quantitation of microbial nucleic acids

Nucleic acid amplification reactions offer a great deal of potential for the accurate and sensitive detectionlquantitation of microbial nucleic acids in human body fluids and have, in some cases, an edge over techniques using immunoassays. Successful detections include : nucleic acids from viruses such as HIV, rotaviruses, coxsackieviruses, papilloma viruses, pestiviruses as well as microorganisms in intestinal fluids. The development of techniques for the amplification of nucleic acids from clinical samples represents a major advance in the ability to eetect and characterize infecting viruses. The most widely used nucleic acid amplification technique is the polymerase chain reaction (ref. 1-3). Polymerase chain reactions and similar systems offer a number of important advantages in terms of viral diagnostics (Fig. 1). However, while these reactions have proven to be extremely valuable for the characterization of small numbers of samples, there are a number of technical problems which have limited the wide-scale application of this technique in diagnostic settings. Of particular importance is the occurrence of cross-contamination. The degree of containment available in clinical settings if often sub-optimal. Furthermore, as the methods are applied to additional body fluids, the problem of sample related inhibitors of enzyme-catalyzed amplification becomes more of concern to diagnostic microbiologists. For example, we have found that fecal samples can contain a material which inhibits the amplification of rotaviral RNA (ref. 4 ) . This material, which is probably a heavy metal or other potent enzymic inhibitor, is resistent to phenol or guanidinium denaturation and co-precipitates with RNA in ethanol. While it can be removed by physical separation techniques such as CF-11 chromatography, the need to perform these reactions markedly increases the time involved in sample preparation and likewise increases the possibility of sample contamination. CrossContamination Amplified Products Plasmids Sample DNA Retroviruses Other DNA-Viruses Human Qenome /nterpretation of Resu/ts Natural History Latency Infectivity Fahe Homo/ogy